The long-term goal of this project is to investigate the neurobiological functions of palmitoylation, the reversible modification of proteins with long-chain fatty acids. Palmitoylation has been linked to Huntington's disease, Ras-dependent tumorigenesis, schizophrenia, and nonsyndromic X-linked mental retardation. Palmitoylation also is critical for proteins that mediate neuronal signaling, transmitter release and reuptake, which suggest roles in Parkinson's disease, depression, addiction or stroke. The breakthrough in the field came from the P.l.'s recent studies that cloned and characterized the first palmitoyltransferase (PAT) enzymes. Humans express at least 23 PATs, which are most highly expressed in the nervous system. The functions of individual PATs or classes of PATs are poorly understood because PAT inhibitors have yet to be reported and only one PAT has been knocked out in mice. Accordingly, the long-term goal of this project is to probe the functions of individual PATs and classes of PATs by identifying and characterizing specific inhibitors of these enzymes. The short-term goal is to develop a novel gain of function system suitable for high-throughput screening of PAT inhibitors. This system will use cloned PATs and a recently identified palmitoylated neuronal protein that regulates signaling by G protein-coupled receptors. The unique feature of this protein is that when palmitoylated it localizes to the plasma membrane whereas when unpalmitoylated it localizes to the nucleus. These properties will be used to engineer a cell-based system in which PAT inhibition leads to production of a luminescence signal. The system will be validated extensively by comparing luminescence when cells express wild type versus non-palmitoylated mutant forms of the G protein regulatory protein, and when cells are treated with a palmitate analog that inhibits PATs as well as enzymes involved in fatty acid metabolism. Negative controls will include specific prenylation inhibitors that do not inhibit palmitoylation. Once validated, this system will be delivered to appropriate NIH screening centers for screening small molecule libraries. In addition, this system will be used in house to screen novel palmitate analogs synthesized by a collaborator. Potential lead compounds will be characterized in the future with an extensive set of functional, cell biological and biochemical assays to eliminate false positives, confirm true positives and investigate inhibitor specificity and mechanism.